The present invention pertains to novel endoproteinases involved in the production of cocoa flavor and the DNA coding for them. In particular, the present invention relates to the use of said enzymes for the manufacture of cocoa flavor.
It is known that in processing cacao beans the generation of the typical cocoa flavor requires two stepsxe2x80x94the fermentation step, which includes air-drying of the fermented material and the roasting step. Though roasting seems to be the key stage of obtaining cocoa flavor subjecting non fermented beans to a roasting step does not yield cocoa flavor suggesting that during the fermentation step precursors are produced that are essential for flavor generation (Rohan J. Food Sci. 29 (1964), 456-459).
During fermentation two major activities may be observed. First, the pulp surrounding the beans is degraded by micro-organisms with the sugars contained in the pulp being largely transformed to acids, especially acetic acid (Quesnel et al. J. Sci. Food. Agric. 16 (1965), 441-447; Ostovar and Keeney, J. Food. Sci. 39 (1973), 611-617). The acids then slowly diffuse into the beans and eventually cause an acidification of the cellular material. Second, fermentation also results in a release of peptides exhibiting differing sizes and a generation of a high level of hydrophobic free amino acids. This latter finding led to the hypothesis that proteolysis occurring during the fermentation step is not due to a random protein hydrolysis but seems to be rather based on the activity of specific endoproteinase (Kirchhoff et al., Food Chem 31 (1989), 295-311). This specific mixture of peptides and hydrophobic amino acids is deemed to represent cocoa-specific flavor precursors.
So far in cacao beans several proteolytic enzyme activities have been investigated and checked for their putative role in the formation of cocoa flavor precursors.
An aspartic endoproteinase activity which is optimal at very low pH (pH 3.5) and is inhibited by pepstatin A has been identified. A polypeptide described to have this activity has been isolated and is described to consist of two peptides (29 and 13 kDa) which are deemed to be derived by self-digestion from a 42 kDa pro-peptide (Voigt et al., J. Plant Physiol. 145 (1995), 299-307). The enzyme cleaves protein substrates between hydrophobic amino acid residues to produce oligopeptides with hydrophobic amino acid residues at the ends (Voigt et al., Food Chem. 49 (1994), 173-180). The enzyme accumulates with the vicilin-class (7S) globulin during bean ripening. Throughout germination, its activity remains constant during the first days and does not decrease before the onset of globulin degradation (Voigt et al., J. Plant Physiol. 145 (1995), 299-307).
A cysteine endoproteinase activity had been isolated which is optimal at a pH of 5. This enzymatic activity is believed not to split native storage proteins in ungerminated seeds. Cysteine endoproteinase activity increases during the germination process when degradation of globular storage protein occurs (Biehl et al., Cocoa Research Conference, Salvador, Bahia, Brasil, Nov. 17-23, 1996).
Moreover, a carboxypeptidase activity has been identified which is inhibited by PMSF and thus belongs to the class of serine proteases. It is stable over a broad pH range with a maximum activity at pH 5.8. This enzyme does not degrade native proteins but preferentially splits hydrophobic amino acids from the carboxy-terminus of peptides. Yet, peptides with carboxy-terminal arginine, lysine, or proline residues are seemingly resistant to degradation. The rate of hydrolysis has been found to be not only determined by the carboxy-terminal amino acid as such, but also to be affected by the neighbouring amino acid residue (Bytof et al., Food Chem. 54 (1995), 15-21).
During the second step of cocoa flavor productionxe2x80x94the roasting stepxe2x80x94the oligopeptides and amino acids generated at the stage of fermentation have been found to obviously undergo a Maillard reaction with reducing sugars present eventually producing the substances responsible for the cocoa flavor as such. This hypothesis has been confirmed in an experiment, wherein an oligopeptide fraction isolated after fermentation of cacao beans had been subjected to roasting in the presence of free amino acids and reducing sugars to obtain cocoa flavor (Mohr et al., Fette, Seifen, Anstrichmittel 73 (1971), 515-521 and 78 (1976), 88-95).
Cocoa-specific aroma has also been obtained in an experiment wherein acetone dry powder (AcDP) prepared from unfermented ripe cacao beans was subjected to autolysis at a pH of 5.2 followed by roasting in the presence of reducing sugars. It was conceived that under these conditions preferentially free hydrophobic amino acids and hydrophilic peptides should be generated and the peptide pattern thus obtained was similar to that of extracts from fermented cacao beans. An analysis of free amino acids revealed that Leu, Ala, Phe and Val were the predominant amino acids liberated in fermented beans or autolysis (Voigt et al., Food Chem. 49 (1994), 173-180). In contrast to these findings no cocoa-specific flavor could be detected when AcDP was subjected to autolysis at a pH of as low as 3.5, the pH, at which the known aspartic endoproteinase shows activity. Only few free amino acids were found to be released but a large number of hydrophobic peptides were formed. This may be explained by the aspartic endoproteinase having a high activity at this pH with the carboxypeptidase being substantially inactive under these conditions. When incubating peptides obtained after autolysis of AcDP at a pH of 3.5 with carboxypeptidase A from porcine pancreas at pH 7.5 hydrophobic amino acids were preferentially released. The pattern of free amino acids and peptides was rather similar to that found in fermented cacao beans and in the proteolysis product obtained by autolysis of AcDP at pH 5.2. After roasting of the amino acids and peptides mixture as above, a cocoa aroma could be generated. On the contrary, with a synthetic mixture of free amino acids alone whose composition was similar to the spectrum found in fermented beans cocoa flavor could not be detected after roasting, indicating that both the peptides and the amino acids are important for this purpose (Voigt et al., Food Chem. 49 (1994), 173-180).
Apart from the enzymes also the protein source of the peptides/amino acids seems to be of importance for the generation of cocoa flavor.
During cacao bean fermentation, the percentage reduction of protein concentration observed for vicilin and albumin was 88.8% and 47.4%, respectively (Amin et al., J. Sci. Food Agric. 76 (1998), 123-128). When peptides obtained by proteolysis of the globulin fraction were post-treated with carboxypeptidase, hydrophobic amino acids (Leu, Phe, Ala, Val, Tyr) were preferentially released and a typical cocoa aroma was detected after roasting in the presence of reducing sugars (Voigt et al., Food Chem. 50 (1994), 177-184). In contrary to that, the predominant amino acids released from the albumin-derived peptides were aspartic acid, glutamic acid and asparagine. Furthermore, no cocoa aroma was detected with the albumin fraction. It was therefore concluded that cocoa-specific aroma precursors are preferentially derived from the vicilin-like globulin of cacao bean. Consequently, the mixture of hydrophobic free amino acids and remaining oligopeptides required for the generation of the typical cocoa flavor components seems to be determined by the particular chemical structure of the cacao vicilin-class globulins.
These globulins isolated from cacao beans were also found to be efficiently degraded by pepsin (an aspartic endoproteinase) and chymotrypsin (a serine endoproteinase). Products derived from cacao globulins by successive proteolytic digestion with pepsin and carboxypeptidase A revealed a typical, but less pronounced cocoa aroma upon roasting. No cocoa aroma precursors were generated by degradation of globulins with chymotrypsin and carboxypeptidase A (Voigt et al., Food Chem, 51 (1994), 7-14). Therefore, the specific mixture of oligopeptides and hydrophobic free amino acids required for the formation of the typical cocoa aroma is not only determined by the structure of the protein substrate but also dependent on the specificity of the cacao enzyme cleaving the protein.
In view of the above data a hypothetical model for the generation of the said mixture of peptides and amino acids, i.e. the cocoa flavor precursors, during fermentation had been devised (FIG. 1), wherein in a first step peptides having a hydrophobic amino acid at their end, are formed from storage proteins, which peptides are subsequently further degraded. For splitting off hydrophobic amino acids from peptides formed in a preceding step the above carboxypeptidase activity seems to be involved. Yet, for the stage of producing the said peptides having C-terminal hydrophobic amino acids, the only known enzymatic activity which might be considered in this respect is an aspartic endoproteinase activity related to that mentioned above. It is also possible that the activity mentioned above is the result of different enzyme activities which are still unknown.
Though some aspects of cocoa flavor production have been elucidated there is still a need in the art to fully understand the processes going on, so that the manufacture of cocoa flavor may eventually be optimized.
The present invention discloses ways to improve the formation of cocoa flavor during processing and manufacturing. This is achieved in a first embodiment by providing tow novel aspartic endoproteinases derived from Th. cacao as identified by SEQ ID No 1 and SEQ ID No 2 or variants thereof obtained by substituting, deleting or adding one or more amino acids, an example of which is the protein identified by SEQ ID No 3, such that the enzymatic activity thereof is essentially retained. The aspartic endoproteinases described here (termed TcAP1 and TcAP2 in the following) are capable of cleaving the vicilin-class globulins isolated from cacao beans so that a successive degradation of the peptides by means of carboxypeptidase will result in a mixture of peptides and amino acids that yields a cocoa flavor upon a reaction with reducing sugars, e.g., upon roasting.
According to another embodiment, the present invention provides DNA sequences coding for the respective endoproteinases. The DNA sequences may be derived according to the genetic code from the amino acid sequences as identified under SEQ ID Nos. 1 and 2 considering the wobble hypothesis, optionally taking account codon preferences of specific hosts, in which the DNA sequences shall be expressed. The skilled person may well devise appropriate DNA sequences based on the polypeptide sequences given and his own technical knowledge and understanding. According to a preferred embodiment the DNA sequences are as identified as SEQ ID No 4 (TcAP1) and SEQ ID No 5 (TcAP2), which DNA sequence may be varied by replacing, deleting or adding one or more nucleotides such, that the endoproteinases essentially retain their enzymatic activity.
The DNA sequences may be used for recombinantly preparing the aspartic endoproteinases of the present invention. To this end the DNA sequences are incorporated into a suitable expression vector, such as a plasmid or a viral vector, which comprise the common sequences, such as a promotor, a polylinker for alleviating the cloning of the DNA sequences therein, leader sequences, to direct the polypeptide produced out of the cell. The vectors will be selected based on the requirements of the system used, e.g. for an expression in E. coli the vectors pGEMEX, pUC-derivates, pGEX-2T, pET-derivates, pQE8 may be envisaged, which are widespread in use and are commercially available. As an example aspartic endoproteinases could be expressed into medium or on surface of lactic acid bacteria used in lactic products such as milk or yogurt.
For expressing the endoproteinases in e.g., yeast the vectors pNFF296, pY100, pPIC9K, pPICz and Ycpad1 may be utilized and for expression in animal cells the vectors pKCR, pEFBOS, cDM8 und pCEV4 as well as pSS-derivates (Kay R. et al., Science 236 (1987), 1299-1302) may be used. Moreover, for expressing the endoproteinases in plant cells, especially in cacao, the vector pAL76 or pBin19-derivates may be used and for insect cells e.g. the vector pAcSGNT-A.
The aspartic endoproteinases may be expressed in a prokaryotic or eukaryotic cell as mentioned above. It will be appreciated that the skilled person will be able to select, based on the need and his own technical skill, an appropriate expression system to achieve the desired goal. In case the endoproteinase shall simply be added to a protein mixture, such as isolated cacao vicilin-class globulins, the recombinant enzyme may be produced in a bacterial system such as E. coli or in yeast and applied on the protein material.
Yet, in view of the implication to increase the enzymatic activity in cacao itself a transgenic plant cell may be envisaged, wherein one or more copies of the endoproteinases, optionally coupled with a suitable and controllable promotor, have been incorporated into the genome of the plant cell. The introduction of the DNA sequence(s) may be achieved by e.g., homologous recombination of DNA stretches harboring one or more copies of the DNA sequences coding for the endoproteinases of the present invention into embryogenic calli prepared beforehand. Since plant cells are totipotent a new transgenic cacao tree may be produced in this way the beans of which will exhibit more rapid degradation of the vicilin-class globulins when subjected to conditions of fermentation.
In consequence a transgenic plant, harboring one or more additional copy(ies) of a DNA sequence coding for the endoproteinases of the present invention is well within the scope of the present invention.
The present endoproteinases may also be used for the manufacture of cocoa flavor by treating a suitable starting material (cacao bean, liquor or crumb), preferably vicilin-class globulins, with said endoproteinases of the present invention and concurrently or afterwards treating the material with carboxypeptidase to obtain a mixture of peptides and amino acids appropriate to act as cocoa flavor precursors. This mixture may then be subjected to xe2x80x9ca roasting stepxe2x80x9d, i.e. may be subjected to a reaction with reducing sugars to eventually obtain cocoa flavor.
Since some of the enzymes involved in the generation of cocoa flavor are now at hand cocoa flavor may be produced artificially without having to rely on the common process of fermenting and roasting cacao beans. The present invention therefore also provides a method for generating cocoa flavor which comprises the step of subjecting a material suitable to yield cocoa flavor precursors, such as the known vicilin-class globulins, to an enzymatic degradation involving the use of the aspartic endoproteinases of the present invention.
In particular, the present aspartic endoproteinases may be overexpressed in protein bodies of plant cells, especially seed cells, and then hydrolysis of the cellular protein material may be effected by treating such plant cells with an acidic solution.
The present endoproteinases may also be used for hydrolyzing proteins by contacting a material of choice, such as the protein in isolated form or material containing the protein, such as e.g. food material, with an endoproteinase of the present invention and effecting hydrolysis to a desired degree. Examples for materials are dairy substances (whey protein, and casein), wheat gluten, corn gluten, meat, egg protein and other protein containing vegetable substances not mentioned above such as proteins from oil seeds, including soybean protein and defatted soy protein.